Julie M. Old

Development of the immune system and immunological protection in marsupial pouch young.

At birth the tissues of marsupial immune system are underdeveloped. The young animal is not immunocompetent. Histological and immunohistochemical studies of pouch young epithelial tissues provide a clear picture of tissue development but the timing of onset of immunocompetence awaits definition. The survival of the neonatal marsupial in a microbially rich environment is dependent on maternal strategies, including immunoglobulin transfer via milk and, in some species, prenatally via the yolk sac placenta. It is also likely that pouch secretions play a role. This review summarizes our current knowledge of the pathway of immunological development in marsupials and the protection and threats afforded by the pouch environment.

The effect of oestrus and the presence of pouch young on aerobic bacteria isolated from the pouch of the tammar wallaby, Macropus eugenii

Qualitative changes in the culturable, aerobic bacterial flora isolated from the tammar pouch have been documented over the period leading up to oestrus, at the time of anticipated birth and in absence and presence of pouch young of varying ages. In a group of 12 animals studied thirty species of aerobic bacteria were isolated. Twenty five species were found in pouches with no pouch young, 9 in pouches with young less than 3 weeks of age and 9 in pouches containing older animals. Gram positive organisms including Corynebacterium spp., Micrococcus spp. and Staphylococcus spp. were the most frequent isolates, regardless of reproductive status. Whilst Gram-negative rods Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterobacter aerogenes, and Escherichia coli were found in pouches with and without pouch young, but not in pouches close to oestrus, at the time of birth or in the presence of very young animals (< 6 days). While pouches without pouch young displayed the greatest diversity of bacterial species, there were still significant numbers of bacterial species in those containing pouch young. Results suggest that the microbial population of the tammar pouch does not seriously compromise the well-being of the young animal during crucial stages of immunological development. The data are consistent with observations on the microflora of the quokka pouch but not with data from the koala.

The detection of mature T- and B-cells during development of the lymphoid tissues of the tammar wallaby (Macropus eugenii)

The distribution of T‐ and B‐cells in the developing lymphoid and immunohaematopoietic tissues of the tammar wallaby were investigated using antibodies to the mature cell surface markers, CD3, CD5 and CD79b. In the thymus, CD3‐ and CD5‐positive T‐cells were first observed at day 12 postpartum whilst rare B‐cells were first detected at day 23. Both T‐ and B‐lymphocytes were first stained on day 21 postpartum in the spleen and day 24 in lymph nodes. In one sample from a 7‐day‐old animal, rare CD79b‐positive (CD79b+) lymphocytes were observed in the gut‐associated lymphoid tissues. However, CD3+ cells were not apparent until day 12 and CD5+ cells were not detected until day 74 postpartum. No lymphocytes were detected in liver or bone marrow samples and no bronchus‐associated lymphoid tissues were observed. The pattern of development and the distribution of T‐ and B‐cells in the lymphoid and immunohaematopoietic tissues were similar to those observed in eutherian mammals and in limited studies of other metatherians. However, the detection of apparently mature T‐ and B‐cells in the thymus and gut‐associated lymphoid tissues (GALT) at the same postnatal age highlights the need for a more substantial study of the development of GALT. This is, at present, limited by availability of marsupial‐specific antibodies.

Immunohistochemistry of the lymphoid tissues of the tammar wallaby, Macropus eugenii

The lymphoid tissues of the metatherian mammal, the adult tammar wallaby, Macropus eugenii, were investigated using immunohistochemical techniques. Five cross‐reactive antibodies previously shown to recognize surface markers in marsupial tissues and five previously untested antibodies were used. The distribution of T‐cells in the tissue beds of spleen, lymph node, thymus, gut‐associated lymphoid tissue (GALT) and bronchus‐associated lymphoid tissue (BALT) was documented using antibodies to CD3 and CD5. Similarly, B‐cells were identified in the same tissues using anti‐CD79b. Antibodies to CD8, CD31, CD79a and CD68 failed to recognize cells in these tissue beds. In general the pattern of cellular distribution identified using these antibodies was similar to that observed in other marsupial and eutherian lymphoid tissues. This study provides further information on the commonality of lymphoid tissue structure in the two major groups of extant mammals, metatherians and eutherians.

Embryonic communication in the nest: metabolic responses of reptilian embryos to developmental rates of siblings

Incubation temperature affects developmental rates and defines many phenotypes and fitness characteristics of reptilian embryos. In turtles, eggs are deposited in layers within the nest, such that thermal gradients create independent developmental conditions for each egg. Despite differences in developmental rate, several studies have revealed unexpected synchronicity in hatching, however, the mechanisms through which synchrony are achieved may be different between species. Here, we examine the phenomenon of synchronous hatching in turtles by assessing proximate mechanisms in an Australian freshwater turtle (Emydura macquarii). We tested whether embryos hatch prematurely or developmentally compensate in response to more advanced embryos in a clutch. We established developmental asynchrony within a clutch of turtle eggs and assessed both metabolic and heart rates throughout incubation in constant and fluctuating temperatures. Turtles appeared to hatch at similar developmental stages, with less-developed embryos in experimental groups responding to the presence of more developed eggs in a clutch by increasing both metabolic and heart rates. Early hatching did not appear to reduce neuromuscular ability at hatching. These results support developmental adjustment mechanisms of the ‘catch-up hypothesis’ for synchronous hatching in E. macquarii and implies some level of embryo–embryo communication. The group environment of a nest strongly supports the development of adaptive communication mechanisms between siblings and the evolution of environmentally cued hatching.

Molecular and morphological description of a Hepatozoon species in reptiles and their ticks in the Northern Territory, Australia.

Abstract Ticks, representing 3 species of Amblyomma, were collected from the water python (Liasis fuscus) and 3 additional reptile species in the Northern Territory, Australia, and tested for the presence of Hepatozoon sp., the most common blood parasites of snakes. In addition, blood smears were collected from 5 reptiles, including the water python, and examined for the presence of the parasite. Hepatozoon sp. DNA was detected in all tick and reptile species, with 57.7% of tick samples (n = 187) and 35.6% of blood smears (n = 35) showing evidence of infection. Phylogenetic analysis of the 18S rRNA gene demonstrated that half of the sequences obtained from positive tick samples matched closest with a Hepatozoon species previously identified in the water python population. The remaining sequences were found to be more closely related to mammalian and amphibian Hepatozoon species. This study confirms that species of Amblyomma harbor DNA of the same Hepatozoon species detected in the water pythons. The detection of an additional genotype suggests the ticks may be exposed to 2 Hepatozoon species, providing further opportunity to study multiple host–vector–parasite relationships.

The appearance and distribution of mature T and B cells in the developing immune tissues of the stripe-faced dunnart (Sminthopsis macroura)

This paper describes the initial appearance and distribution of mature T and B cells in the developing immune tissues of the stripe‐faced dunnart (Sminthopsis macroura) based on the use of species cross‐reactive antibodies to the lymphocyte cell surface markers CD3, CD5 and CD79b. At birth no mature T or B cells were detected in the liver or bone marrow using anti‐CD3, anti‐CD5 or anti‐CD79b antibodies. T cells were detected in the thymus with anti‐CD3 by day 12 and anti‐CD5 by day 50 postpartum, and T cells in the spleen were detected by day 43 and day 80 postpartum using anti‐CD3 and anti‐CD5, respectively. B cells were observed in the dunnart spleen by 43 days after birth. CD3‐ and CD79b‐positive cells were detected in the lymph nodes by 50 days and CD5 by day 15 after birth, and in the gut‐associated lymphoid tissues by day 50 and anti‐CD5 by day 57 postpartum. The development and distribution of T and B cells in the immune tissues of dunnart pouch young is similar to that described in other marsupial species. Low numbers or absence of mature lymphocytes in immune tissues of early pouch young dunnarts further support the proposition that young marsupials are reliant on non‐specific defence strategies and/or maternal strategies for a significant period of their time of development in the pouch.

Histology and immunohistochemistry of the gut-associated lymphoid tissue of the eastern grey kangaroo, Macropus giganteus

Mesenteric lymph nodes and gut‐associated lymphoid tissue (GALT) from juvenile eastern grey kangaroos were investigated. The mesenteric nodes had a similar structure to that described for eutherian mammals. They contained distinct regions of medulla and cortex, with prominent follicles and germinal centres. Gut associated lymphoid tissue consisted of areas of submucosal follicles. These varied from areas of densely packed lymphocytes with darkly staining, prominent coronas to areas with no defined follicles. The distribution of T cells in these tissues was documented by use of species‐crossreactive antibodies to the surface markers CD3 and CD5; B cells were identified by antibodies to CD79b. Within the lymph nodes T cells were located mainly in the paracortex and cortex, with limited numbers observed in the follicles; B cells were located on the marginal zone of the follicles. In GALT, T cells were located in the peripheral regions of the germinal centres of secondary follicles, while B cells were abundant in primary follicles. These observations are consistent with those made in a range of other marsupials (metatherian) and eutherian mammals and are indicative of the capacity to respond to antigens entering via the mouth.

Detection of a Hepatozoon and spotted fever group Rickettsia species in the common marsupial tick (Ixodes tasmani) collected from wild Tasmanian devils (Sarcophilus harrisii), Tasmania

Tasmanian devils are the largest extant marsupial carnivores, confined to the Australian island state of Tasmania. The iconic marsupial has dramatically declined in number since the discovery of devil facial tumor disease in 1996 and efforts are being made to uncover vital information to assist in the long-term survival of the species. Ticks are the main vectors of arthropod-borne disease in animals, raising the question of whether Tasmanian devils may be host to arthropods capable of harboring infectious agents. Partially engorged ticks were collected from 35 wild Tasmanian devils and tested for the presence of a range of tick-borne genera. A spotted fever group Rickettsia was detected in 45.5% of samples of the tick Ixodes tasmani (n=44), from all trapping locations, sharing close sequence identity to members of the Rickettsia massiliae group. A Hepatozoon species was also identified in 34.1% of the same sample set, sharing sequence similarities to Hepatozoon felis, a known pathogen of felids. Dual detection was identified in 13.6% of tick samples, where prevalence of the two genera overlapped. The existence of two previously undetected species of genera known for containing pathogens identifies additional potential risks to the health of the devil population.

A Histological Investigation of the Lymphoid and Immunohaematopoietic Tissues of the Adult Stripe-Faced Dunnart (Sminthopsis macroura)

This is the first published description of the lymphoid and immunohaematopoietic tissues of an Australian polyprotodont, the stripe-faced dunnart, Sminthopsis macroura and the first account of bronchus-associated lymphoid tissue (BALT) in a metatherian. Histologically, the tissue beds are similar in appearance to those reported in other adult eutherian and metatherian mammals. The liver and bone marrow were mature and virtually no haematopoietic activity was observed. The thymus had undergone involution but retained some lymphocytes. The spleen was similar to that observed in other metatherians containing areas of red and white pulp separated by a marginal zone. Lymph nodes, except for a pair in the posterior abdomen, were difficult to locate but were similar to those observed previously in other adult metatherians. Peyer’s patches were present; however, they lacked dome regions and sometimes had villi above them. BALT appeared to be both compartmentalised and non-compartmentalised in the adult stripe-faced dunnart.